Occurrence of Alternaria solani-nigri Causing Leaf Spot on Cone Pepper in China

Cone pepper (Capsicum annuum var. conoides) is one of the most economically important vegetable globally (Wang et al. 2023). It is an important cash crop in southern China. In August of 2024, pepper leaf spot symptoms were observed in Jiaolong Village (25°90′N, 114°27′E), Shangyu County, Jiangxi Province, China, with an incidence rate of approximately 40% in a cone pepper plantation. Diseased leaves initially developed round brown necrotic spots surrounded by a yellow halo with a white central dot, finally progressing to chlorosis and senescence. Samples of disease leaves were collected, and sliced into small pieces, followed by being sterilized in 75% ethanol for 30 s and 1 % NaOCl for 2 min, and rinsed in sterile distilled water (SDW) three times. The tissue pieces were cultured onto potato dextrose agar (PDA) at 25℃ in darkness for 3-5 days. Eight isolates were obtained, and all showed the same morphological characteristics. The colonies on PDA were circular, white to smoke grey in the center, greyish sepia at the edge. Conidial morphology was determined on potato carrot agar (PCA) and V8 juice agar (V8A) under an 8/16h light/dark cycle (Simmons 2007). Conidia were cylindrical to obovate (38.3-61.2×8.1-13.3 µm on PCA and 41.2-86.6×9.9-19.1 µm on V8A), solitary, 1-3 transverse septa, and beaks distinct and filiform (59.6-123.1×1.3-2.7 µm on PCA and 39.3-109.9×1.9-3 µm on V8A). Based on morphological characteristics, the pathogen was identified as Alternaria solani-nirgi (Simmons 2007; Dubey et al. 1999), with synonyms including A. ascaloniae, A. beticola, A. cyphomandrae, A. glyceriae, A. herbiculinae. (Woudenberg et al. 2014). DNA sequences obtained for strains YzU 241824 and YzU 242154, including RNA polymerase second largest subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1 alpha (TEF1), and internal transcribed spacer (ITS), were amplified with paired primers RPB2-5F/RPB2-7cR (Liu er al. 1999), GPD1/GPD2 (Berbee et al. 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), and ITS4/ITS5 (White et al. 1990), respectively. The resulting sequences were deposited in GenBank (YzU 241824: PV578773 for RPB2, PV578775 for GAPDH, PV578774 for TEF1, PV577193 for ITS). A phylogenetic tree was constructed based on these sequences, using the Maximum Likelihood (ML) method with 1000 bootstrap (BS) replicates and the GTRCAT model in RAxML v.7.2.8 software. The present strains clustered with strain A. solani-nigri (representative strain of CBS 113403) supported with 98% bootstrap values. Mycelial agar plugs (6 mm diameter) from PDA were inoculated onto leaves (n=3) of two-month-old pepper seedlings (C. annuum var. conoides and Capsicum frutescens, n=3). The controls were inoculated with empty PDA plugs. The plants were maintained at 25°C with humidity levels over 85% in a greenhouse. The same assay was performed in triplicate to enhance data validity. After seven days, inoculated leaves showed symptoms consistent with those in the field, whereas the control remained asymptomatic. There-isolation was verified via morphology and the RPB2 gene sequences, thereby fulfilling Koch's postulates. A. solani-nigri was reported to cause leaf spots on Solanum nigrum, Allium ascalonicum, Glyceria maxima, and Beta vulgaris (Woudenberg et al. 2014). To our knowledge, this is the first report of pepper leaf spot caused by A. solani-nigri in China, which will provide a theoretical basis for the management of pepper diseases.